Allergenic risk assessment of

insect proteins

Kitty Verhoeckx

Alternative sustainable protein sources

Insects (May 2013: report FAO on edible insects)

Refining waste streams (e.g. beet leaves)

Algae, duckweed, seaweed (no agricultural land required)

Engineered/ processed proteins in food industry (e.g. GMO crops, hydrolysed wheat-proteins)

General food law (EC regulation No 258/97 and EU recommendation 97/618)

• The law requires that safety is assured for all

food ingredients placed on the market.

• Responsibility of the producers

• Novel food law: Comprehensive food safety

assessment for novel foods introduced after 1997

Guidance novel foods 21 Sep 2016,(amending EC regulation No 97/618 and 2015/2283)

Regulation is in force since January 2018

Default assumption for Novel Foods containing proteins is that such

Novel Foods have allergenic potential

Comprehensive literature review in order to retrieve available

information on sensitisation, and on case reports of allergic reactions

and/or allergenicity studies (in vitro, in animals, in humans) of the

Novel Food and/or its source(s).

Draft guidance novel foods(amending EC regulation No 97/618 and 2015/2283)

Protein analysis

Protein content in the Novel Food

Immunological tests (e.g. Western blotting)

Molecular weight of the potentially allergenic protein

heat stability, sensitivity to pH, digestibility by gastrointestinal

proteases,

Degree of sequence homology with known allergens.

Human testing

Detection of specific IgE antibodies

Skin prick testing

Double blind placebo controlled food challenge studies

• Based on GMO and useful for

single proteins

✓ The mealworm is the larval stage of the Yellow mealworm

beetle

✓ Originally produced as feed for animals such as fish, reptiles

and birds.

✓ Commercially available for human consumption

Allergenicity assessment of a novel food:

How to screen for potential new Yellow Mealworm

(Tenebrio Molitor) allergens?

New protein (source)

New protein (source)

History of human exposure (work/food)

Relationship: Taxonomy, Homology

Identification proteins in source

Information on usage: raw, matrix, processing

Research material:Extract(s), GMP

Research protocol, METC

Cross reactivitySera known allergy with new protein

Functional Cross reactivity

Sera known allergy with new protein

Sensitising potency

• Immuno-blot• ELISA• CAP/ISAC

Skin prick test

Provocation

History SensitisationScreening (interview and/or serology )

• Working population • Targeted people with symptoms

No history

New allergy Sera new allergy with new protein• In vitro• In vivo

Cross reaction Sera new allergy with known allergen• In vitro• In vivo

Identification proteins

Identification reactive proteins in source(potential new allergens)

New allergiesCross reactivityNew allergies & Cross reactivity

Basophil activation test

Verhoeckx et al 2016, Regul Toxicol Pharmacol

History of Allergic reactions

History of human exposure

(work/food)

✓Allergic reactions to insects bites

✓Allergic reactions caused by inhalation (e.g.

dust with cockroach feces) and contact (e.g.

caterpillar hairs) and primarily occur with people

who regularly come into contact with insects

(e.g. entomologists, fish bait breeders)

✓A number of cases have also been documented

in which the consumption of insects has

caused an allergic reaction and even

anaphylactic shock in humans

Mealworms are closely related to shrimp

and house dust mite.

Relationship:

Taxonomy, Homology

Domain EukaryotaDomain bacteria Domain protozoa

Life

Kingdom Animalia

Verhoeckx & van Broekhoven Food and Chemical Toxicology (2014)

Mealworm proteins identified with LC-MS/MS

Verhoeckx & van Broekhoven Food and Chemical Toxicology (2014)

Identification proteins in source

Tris(Extract 1 and 2) Urea (Extract 3)

Calcium-transporting ATPase sarcoplasmic

Myosin heavy chain

Arginine kinase Actin

Actin Larval cuticle protein

POTE ankyrin domain family member F Late histone H2A

Tubulin alpha-1 Tropomyosin-1

Catalase Pupal cuticle protein G1A

Alpha-amylase Ovalbumin-like

Muscle-specific protein 20 Tropomyosin-2

Glyceraldehyde-3-phosphate dehydrogenase

Myosin-2

Ovalbumin-like ATP synthase subunit beta OS

Tubulin beta chain (Fragment) Fructose-1,6-bisphosphatase

class 3

Elongation factor 2 Troponin T

V-type proton ATPase catalytic subunit A

Sarcoplasmic Ca binding protein

Identification based on sequence

homology with Drosophila melanogaster

208 Mealworm proteins tested with Allermatch

Relationship:

Homology

(Bioinformatics)

• Cross reactivity only with mites.

• Topomyosin sequence not available for mealworm

New protein (source)

Relationship: Taxonomy, Homology

Identification proteins in source

Information on usage: raw, matrix, processing

Research material:Extract(s), GMP

Research protocol, METC

Cross reactivitySera known allergy with new protein

Functional Cross reactivity

Sera known allergy with new protein

• Immuno-blot• ELISA• CAP/ISAC

Identification proteins

Cross reactivity

Basophil activation test

History of human exposure (work/food)

Cross reactivity

Skin prick test

Provocation

Cross reactivity

Functional cross reactivity

Sera known allergy with mealworm

Cross reactivitySera known allergy

with mealworm

2 3

Patient 2 3

Extract 1

Extract 2

Extract 3

Patient 2

Patient 1

Patient 1

Patient 3

Patient 3

Verhoeckx & van Broekhoven Food and Chemical Toxicology (2014)

Immuno blot

BAT

Tropomyosin Arginine kinase

Clinical study: Functional cross-reactivity with

Shrimp allergic patients

Shrimp allergic patients

Characterisation patients

Clinical signs (Questionnaire)

Skin Prick Test (SPT)

Serology (CAP, ISAC)

Basophil Activation Test (BAT)

Immuno-blot (blot)

Food challenge with mealworm snack (n = 15)

Broekman et al 2016 JACI

13 of 15 shrimp allergic patients react to mealworm in a food challenge

PatientSex

(Male/ Female)

Age (years) 0.01 g

(2,16 mg)

0.1 g (21,6 mg)

1 g (216 mg)

3 g (648 mg)

10 g (2,16 g)

30 g (6,48 g)

60 g (12,96 g)

Mealworm challenge

Muller

A F 46OA, S, GI 2

B F 23 OA,S,R OA,S,GI OA, S, GI,R 3

C M 69 OA OA OA 0

D M 45 S, GI 2

E F 27 OA OA OA, S 1

F M 19 S GI 2

G F 60 S S 1

H M 30 GI 2

I M 27 Neg

J F 47 S S, R S, GI,R 3

K F 52 Neg

L M 26 S, GI 2

M M 34 OA OA OA 0

N F 23OA, S OA OA OA, S 1

O M 46 OA OA OA OA,GI 2

OA Oral allergy, local S Skin/mucosa GI Gastrointestinal R Respiratory C Cardiovascular Dose not given

Provocation

Are house dust mite patients at risk

when eating mealworm?

Cross reactivity

Sera known allergy

with new protein

Bioinformatics HDM allergens: (BLAST

Uniprot)

HDM mealworm overlap

Der p 1 C1 family cathepsin L15

(Tenebrio molitor)

32.7%

Der p 2 No alignment

Der p 23 No alignment

Der p 23 has shown homology to, so far not

completely characterized allergens from

cockroach.

Bioinformatics HDM allergens:

(AllergenOnline)

C1 family cathepsin L15 (Tenebrio molitor)

HitDefline

Best # Hits Full Alignment

# %ID > 35% E-val %ID length

12 Der f 1 allergen 47.50% 66of247 1.2e-029 31.50% 330

23 Der p 1 allergen preproenzyme 46.30% 87of247 1.7e-029 32.70% 281

80mer Sliding Window Search Results

• C1 family cathepsin L15 protein could be one of

the cross reactive proteins for HDM and mealworm

208 Mealworm proteins tested with Allermatch

Sensitisation

Clinical study: Primary mealworm allergy

People with allergic complaints for mealworm (n=4) (e.g. insect

farmers). Clinical symptoms of inhalant (n=2) and food allergy

(n=2) to mealworm

Characterisation patients (n=4)

Clinical signs

Skin Prick Test (SPT)

Serology

Basophil Activation Test (BAT)

Immuno-blot

Food Challenge with mealworm snacks (n=4)

Food Challenge with shrimp (n=4)Broekman et al J Allergy Clin Immunol. 2017

Results de novo sensitisation

All subjects (n=4) were sensitized to mealworm according to all

tests.

Two subjects had a positive DBPCFC to mealworm, but were

not allergic to shrimp.

Responsible allergen: Larval Cuticle protein.

De novo sensitisation to mealworm is possible.

New protein (source)

History of human exposure (work/food)

Relationship: Taxonomy, Homology

Identification proteins in source

Information on usage: raw, matrix, processing

Research material:Extract(s), GMP

Research protocol, METC

Sensitising potency

No history

New allergiesNew allergies & Cross reactivity

Sensitisation

Aim: To build an interdisciplinary European network

of scientists with a broad range of expertise to discuss,

with an out-of-the-box view, new ideas and more

predictive models and approaches to improve the

current allergenicity risk assessment strategy

www.imparas.eu

Random Forrest model: Tanja Krone

Can bioinformatics act as a confirmation

of newly discovered allergens in food?

Traditional – FASTA algorithm used with novel proteins to

identify source organism and similarity with known allergens

New - Random Forrest model: machine learning tool based on

physical-chemical properties

Improved bioinformatic approach working towards predictive

bioinformatics?

Results FASTA3 program package Input Sequence Name: sp|P80681_Larval_cuticle_A1A, (Allergen 2015 - FASTA 3.45 )

Database Match Description 8mer Match Species %Identity Overlap EValue

Der p 7 allergen-like n/a

Dermatophagoides

pteronyssinus 30,1 73 3,50E+00

Der p 7 allergen polypeptide n/a

Dermatophagoides

pteronyssinus 28,8 73 7,30E+00

Phlp5 n/a Phleum pratense 27,2 114 3,60E+00

Pollen allergen KBG 41 precurso n/a 35,5 62 6,30E-02

Pollen allergen KBG 60 precurso n/a 33,9 62 1,10E+00

arginine kinase n/a Scylla paramamosain 35,6 59 7,20E+00

group V allergen Phl p 5.0203 precursor n/a Phleum pratense 31,7 63 8,30E+00

group V allergen Phl p 5.0206 precursor n/a Phleum pratense 31,7 63 8,10E+00

group V allergen Phl p 5.0207 precursor n/a Phleum pratense 31,7 63 8,10E+00

• No significant homology with

known allergenic proteins!

Results Random Forrest model

Name species Predicted allergen Allergenic

Larval cuticle protein A2B Tenebrio molitor Y Y

Larval cuticle protein A1A Tenebrio molitor Y Y

Larval cuticle protein A3A Tenebrio molitor Y Y

Alpha-amylase Tenebrio molitor Y Y

Tropomyosin-1, isoforms 9A/A/B Drosophila melanogaster Y Y

Arginine kinase Drosophila melanogaster Y Y

Cytochrome b Tenebrio molitor N N

Elongation of very long chain fatty acids protein Tenebrio molitor N N

• Larval cuticle proteins identified as allergenic

protein!

Bioinformatics – The Larval Cuticle

proteins:

FASTA3 program package from W.R. Pearson : sequence

alignment/Sequence homology

Important use of bioinformatics in conjunction with qualified allergen

database can work well in many cases, but can miss undiscovered,

new structural classes of proteins if protein not closely related to

known allergens.

Random Forest – New science developed as part of Shared

Research Program @ TNO.

In this work, RF was not available for the beginning of the

mealworm studies, but nicely confirms the work and would have

given some good direction if available at the beginning of process –

appears to function better at prediction.

Take home messages:Shrimp and HDM patients are possibly at risk when eating

insects, (based on cross reactivity).

De novo sensitization to mealworm is possible.

Bioinformatics in combination with history and taxonomy

makes is a powerful process in investigating food allergy.

Bioinformatic tools can be used to identify potential cross-

reactivity, however protein sequences must be available and

batch acquisition possible!!

Improved prediction and validation are needed to help

identify new classes of protein allergens

There are no bioinformatic tools for predicting de novo

sensitization and new allergies, but Random Forest looks

promising.

Thank you for your attention

Dr. Kitty Verhoeckx

Scientist, TNO

T: +31 (0)88 8665136

@: Kitty.verhoeckx@tno.nl

SRP TNO www.srpfoodallergy.com

COST Action ImpARAS www.imparas.eu

Acknowledgements

Henrike Broekman

André Knulst

Stans den Hartog Jager

Eelco Botter

Jan Ruig

Margot Calis

Marieke Calis

Marian Peters

Lucia Jimeno Nogales

Marco Gaspari

Aard de Jong

Astrid Kruizinga

Geert Houben

Tanja Krone

Greg Ladics

Lilia Babe

Scott McClain